© 2016. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2016) 219, 2060-2065 doi:10.1242/jeb.137190
RESEARCH ARTICLE
The ability to survive intracellular freezing in nematodes is related
to the pattern and distribution of ice formed
ABSTRACT
A few species of nematodes can survive extensive intracellular
freezing throughout all their tissues, an event that is usually thought
to be fatal to cells. How are they able to survive in this remarkable
way? The pattern and distribution of ice formed, after freezing at
−10°C, can be observed using freeze substitution and transmission
electron microscopy, which preserves the former position of ice as
white spaces. We compared the pattern and distribution of ice
formed in a nematode that survives intracellular freezing well
(Panagrolaimus sp. DAW1), one that survives poorly (Panagrellus
redivivus) and one with intermediate levels of survival (Plectus
murrayi). We also examined Panagrolaimus sp. in which the survival
of freezing had been compromised by starvation. Levels of survival
were as expected and the use of vital dyes indicated cellular
damage in those that survived poorly (starved Panagrolaimus sp.
and P. murrayi). In fed Panagrolaimus sp. the intracellular ice
spaces were small and uniform, whereas in P. redivivus and starved
Panagrolaimus sp. there were some large spaces that may be
causing cellular damage. The pattern and distribution of ice formed
was different in P. murrayi, with a greater number of individuals
having no ice or only small intracellular ice spaces. Control of the
size of the ice formed is thus important for the survival of
intracellular freezing in nematodes.
KEY WORDS: Intracellular ice, Panagrolaimus, Antarctic, Freeze
substitution, Transmission electron microscopy
INTRODUCTION
Freezing-tolerant animals can survive ice forming in their bodies
(Lee, 2010). They include some invertebrates (e.g. insects,
nematodes, molluscs) and ectothermic vertebrates (e.g. frogs,
hatchling turtles). Intracellular ice formation is usually thought to
be fatal because mechanical disruption may be caused by the
expansion of water as it freezes, the puncturing of membranes by ice
crystals and the redistribution (recrystallization) of ice crystals after
freezing and during thawing (Acker and McGann, 2001; Karlsson
et al., 1993; Muldrew et al., 2004). There are, however, examples of
particular cells and tissues of insects (e.g. fat body and midgut cells)
surviving intracellular freezing, and this may be widespread in
freezing-tolerant insects (Sinclair and Renault, 2010). Even some
isolated mammalian cells and tissues will survive intracellular
freezing (Acker and McGann, 2002; Rall et al., 1980; Sherman,
Department of Zoology, University of Otago, P.O. Box 56, Dunedin 9054, New
Zealand.
*Present address: GBIF Secretariat, Universitetsparken 15, Copenhagen DK-2100,
Denmark.
‡
Author for correspondence ([email protected])
D.A.W., 0000-0001-6369-4984
Received 14 January 2016; Accepted 27 April 2016
2060
1962) and the frozen cells may even survive better than unfrozen
cells (Acker and McGann, 2002).
The only animals that have been shown to survive extensive
intracellular freezing throughout their tissues are a few species of
nematodes, particularly Panagrolaimus davidi Timm 1971
(Wharton and Ferns, 1995). The species used in this study is now
known as Panagrolaimus sp. DAW1, as rRNA sequencing shows
that field-sourced P. davidi and the cultured strain are different
species (Raymond et al., 2014). Panagrolaimus davidi (field strain)
is found in coastal areas in the McMurdo Sound region of Antarctica
(Wharton and Brown, 1989), particularly in soil supplied with
liquid water close to bird colonies (Raymond and Wharton, 2013),
whereas Panagrolaimus sp. DAW1 has been isolated from similar
habitats at Cape Bird and Cape Royds, Ross Island, Antarctica
(D.A.W., unpublished observations). Soil surface temperatures as
low as −39.6°C have been recorded at Cape Bird (Sinclair and
Sjursen, 2001). Some other species of nematodes have been shown
to have a limited ability to survive intracellular freezing (Ali and
Wharton, 2014; Wharton and Raymond, 2015).
The ability of Panagrolaimus sp. DAW1 to survive intracellular
freezing is dependent upon its nutritional state (Raymond and
Wharton, 2013). This provides the opportunity to make comparisons
between fed cultures with a high level of survival of this stressor and
starved cultures with a lower level of survival. Freeze substitution
preserves the former position of ice in frozen nematodes and the
pattern and distribution of ice formed can be observed by
transmission electron microscopy (Wharton et al., 2005). In the
present study, we have compared the pattern and distribution of ice
formed in fed and starved Panagrolaimus sp. after freezing at a
temperature (−10°C) that produces intracellular freezing (Raymond
and Wharton, 2013) and used vital dyes (Lee et al., 1993) to look for
cellular damage. We have also made comparisons with the oatmeal
nematode, Panagrellus redivivus (Linnaeus 1767) Goodey 1945,
whose natural habitat is thought to be fermenting tree resin, has very
low survival after freezing at −10°C (Hayashi and Wharton, 2011);
and with another Antarctic nematode with a similar distribution to
that of P. davidi, Plectus murrayi Yeates 1970, that has intermediate
levels of survival (Raymond et al., 2013; Wharton and Raymond,
2015). We used nematodes grown at 20°C to avoid any starvation
effects of cold acclimation in our fed cultures (Raymond and
Wharton, 2013). Our hypothesis is that the pattern and distribution
of ice formed will reflect the different levels of survival of
intracellular freezing between these treatments and species.
MATERIALS AND METHODS
Panagrolaimus sp. DAW1 (formerly known as Panagrolaimus
davidi or P. davidi CB1) is available from the Caenorhabditis
Genetics Centre (www.cbs.umn.edu/research/resources/cgc). Fed
and starved cultures were generated as described by Raymond and
Wharton (2013). Plectus murrayi were isolated from the Victoria
Land coast at Terra Nova Bay Antarctica and cultured on 0.1%
Journal of Experimental Biology
Mé liane R. Raymond* and David A. Wharton‡
Survival of a stress that produces intracellular freezing
Survival of a freezing stress known to produce intracellular freezing in
Panagrolaimus sp. DAW1 (Raymond and Wharton, 2013) was tested
by transferring subsamples of the cultures used for freeze substitution
(see below) of ∼100 nematodes in 10 µl of ATW to small
microcentrifuge tubes. These were then placed in a cooling block
(Wharton et al., 2004), the temperature of which was controlled by a
programmable refrigerated circulator (Haake Phoenix II, Thermo
Fisher Scientific, Waltham, MA, USA). Sample temperatures were
monitored by thermocouples immersed in the sample and interfaced
to a computer via a Powerlab A/D converter (ADInstuments,
Dunedin, New Zealand). The samples were cooled from +1°C to
−10°C at 0.5°C min−1 and if freezing had not occurred spontaneously
it was initiated by adding a small ice crystal. Samples were held at
−10°C for 30 min, warmed to +1°C at 0.5°C min−1 and then
transferred to a watchglass at room temperature in 1 ml of ATW.
Survival was determined after 24 h by counting the proportion of
nematodes moving after a physical stimulus (stirring with a pipette)
and compared between groups (fed and starved Panagrolaimus sp.,
P. murrayi and P. redivivus) using one-way ANOVA after arcsin
transformation of percentages in SPSS (SPSS Inc., Chicago, IL,
USA). Levene’s test indicated that variances were significantly
different between treatments and so a post hoc test that does not
assume equal variances was used (Tamhane’s T2) to compare means.
Vital staining
Cell survival was tested using vital dyes on nematodes frozen at
−10°C by the regime above after 24 h recovery and on unfrozen
controls. Whole nematodes were stained with propidium iodide and
Acridine Orange using previously described protocols (Lee et al.,
1993; Sinclair and Wharton, 1997), observed by epifluorescence on
a Zeiss Axiophot microscope, and the proportion containing all
green nuclei (alive), all red nuclei (dead) and a mixture of red and
green nuclei was counted.
Freeze substitution and transmission electron microscopy
Concentrated samples of nematodes (100–200 in 10 µl of ATW, four
replicates with each replicate being from a separate culture) were
frozen by the regime described above and were plunged into liquid
nitrogen after completing 30 min at −10°C. They were then
transferred to a freeze substitution mixture (3% glutaraldehyde, 1%
osmium tetroxide and 0.5% uranyl acetate in methanol) at −90°C in a
Reichert automatic freeze substitution apparatus. They were processed
for transmission electron microscopy as described by Wharton et al.
(2005) into Spurr resin in a single conical beam capsule for each
replicate. Sections were cut on a Reichert ultramicrotome, picked up
on Formvar-coated copper grids, stained with uranyl acetate and lead
citrate and observed on a Philips 410 transmission electron
microscope operated at 80 kV. Unfrozen control samples were
processed by conventional chemical fixation (Wharton et al., 2005).
The highly concentrated nature of the sample (100–200
nematodes) ensured that a single section of each replicate
provided enough individuals for analysis. The section was
selected randomly as the first suitable section to be seen under the
microscope (no tears, not too thick). The nematodes within that
section were selected randomly by systematically searching the
section starting at its top left corner, to ensure that no individual was
missed or counted/measured twice. All the nematodes that were
transversely sectioned (indicated by a round rather than an oblique
profile) through the intestinal region were classified as containing no
ice (these often had a shrunken appearance), extracellular ice only or
intracellular ice (these often also had extracellular ice), as described
by Wharton et al. (2005). Differences between species or treatment
in the proportion containing intracellular ice were analysed using
one-way ANOVA as for survival. Levene’s test indicated that
variances were not significantly different between treatments and so
a Scheffe post hoc test was used to compare means.
The section was searched again as before and the first 10
nematode transverse sections seen from each of the four replicates
for each group were photographed for further analysis. The total
body area and the area of the five largest ice-filled spaces were
measured using Image J (Rasband, 1997-2005). The proportional
body area occupied by the five largest ice-filled spaces was
compared using univariate GLM after square root transformation of
percentages. Levene’s test indicated that variances were not
significantly different between treatments and so a Scheffe post
hoc test was used to compare means. There were no significant
differences between replicate cultures and so the data from these
were combined. The proportion of the body area occupied by the
largest ice-filled space and the proportion of nematodes that
contained at least one large ice-filled space (defined as being >10%
of the total body area) were also calculated.
RESULTS
Survival and vital staining
Fed Panagrolaimus sp. DAW1 survived freezing at −10°C best with
79% surviving, followed by starved Panagrolaimus sp. DAW1 and
then P. murrayi; survival was very low (<3%) in P. redivivus
(Fig. 1). Survival was significantly different between the four
groups (F3,12=134.5, P≤0.0001).
100
80
a
60
b
40
b
20
c
0
Panagrolaimus Panagrolaimus
fed
starved
P. murrayi
P. redivivus
Fig. 1. Survival of fed and starved Panagrolaimus sp. DAW1, Plectus
murrayi and Panagrellus redivivus after freezing at −10°C for 30 min. Data
are means ±s.e.m. (N=4, replicate cultures). Different lowercase letters
indicate significant differences between means (P<0.05).
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Journal of Experimental Biology
nutrient agar or sand agar plates (Wharton and Raymond, 2015), with
a layer of artificial tap water (ATW; Greenaway, 1970) or balanced
salt solution (BSS; Piggott et al., 2000). Oatmeal nematodes,
Panagrellus redivivus, were sourced from a commercial supplier
(as ‘microworms’, Biosuppliers, Auckland, New Zealand) and
cultured on autoclaved rolled oats (Hayashi and Wharton, 2011).
Panagrolaimus sp. and P. murrayi are parthenogenetic females,
P. redivivus are mixed males and females. Cultures (mixed stages)
were grown at 20°C for 2 weeks and nematodes were harvested using
a modified Baermann funnel technique (Hooper, 1986). Samples
were taken from four replicate cultures, and the nematodes were
washed three times in ATW or BSS and used immediately for
experiments. This work was performed in accordance with the
procedures of the University of Otago’s Animal Ethics Committee.
Journal of Experimental Biology (2016) 219, 2060-2065 doi:10.1242/jeb.137190
Survival (%)
RESEARCH ARTICLE
RESEARCH ARTICLE
Journal of Experimental Biology (2016) 219, 2060-2065 doi:10.1242/jeb.137190
100
Percentage
80
60
40
20
0
Panagrolaimus
fed
Panagrolaimus
starved
P. murrayi
Fig. 2. Percentage of fed and starved Panagrolaimus sp. DAW1 and
Plectus murrayi that had all green-stained (alive, filled bars), all redstained (dead, open bars) or a mixture of red- and green-stained nuclei
(hatched bars) after freezing at −10°C for 30 min and vital staining. Bars
indicate the mean of two technical replicates.
In unfrozen controls of fed and starved Panagrolaimus sp. DAW1
and of P. murrayi, >90% of nematodes contained all live (greenstained) cell nuclei after vital staining. In P. redivivus unfrozen
A
controls there was no staining of cell nuclei, just green staining of
the lumen of the oesophagus.
Most fed Panagrolaimus sp. DAW1 contained all live (greenstained) cell nuclei after freezing and vital staining (89.4%). In
contrast, only 43% of starved Panagrolaimus sp. DAW1 contained
all live cells after freezing and vital staining; the remainder
contained all dead (red-stained) cells or a mixture of red- and greenstained cells (Fig. 2). In those containing both red- and greenstained cells, dead cells were found most frequently in the head and
middle regions of the body and less frequently in the tail region. All
tissue types (oesophagus, muscle, intestine) contained dead cells,
with damage usually occurring in several tissues.
In P. murrayi after freezing and vital staining, 67.2% of nematodes
contained all live cells, the remainder containing a mixture of greenand red-stained cells. In P. redivivus after freezing and vital staining
there was too much structural disruption to distinguish nuclei, but
both red- and green-stained material was seen.
Pattern and distribution of ice formed
Structural differences can be seen between unfrozen chemically
fixed controls of fed and starved Panagrolaimus sp. DAW1
(Fig. 3A,B). Most noticeable are large spaces filled with lightly
stained granular material that appear in the cytoplasm of
B
mc
il
il
mc
ic
ic
C
D
mc
il
ic
il
Fig. 3. Transmission electron
micrographs of Panagrolaimus sp. and
Panagrellus redivivus.
(A) Panagrolaimus sp. DAW1 fed,
unfrozen, conventional fixation;
(B) Panagrolaimus sp. DAW1 starved,
unfrozen, conventional fixation;
(C) Panagrolaimus sp. DAW1 fed, frozen,
freeze substitution; (D) Panagrolaimus sp.
DAW1 starved, frozen, freeze substitution;
(E) P. redivivus fed, frozen, freeze
substitution; (F) P. murrayi fed, frozen,
freeze substitution. ic, intestinal cell
cytoplasm; il, intestinal lumen; mc, muscle
cell. Scale bars=5 µm. Most of the ice is
intracellular but some areas of
extracellular ice are indicated in C and
F (arrows).
mc
E
F
il
mc
ic
ic
mc
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il
Journal of Experimental Biology
ic
100
a,b
Journal of Experimental Biology (2016) 219, 2060-2065 doi:10.1242/jeb.137190
a
a,b
b
Percentage
80
60
40
20
0
Panagrolaimus Panagrolaimus
fed
starved
P. murrayi
P. redivivus
Fig. 4. Percentage of fed and starved Panagrolaimus sp. DAW1, Plectus
murrayi and Panagrellus redivivus that had intracellular ice (filled bars)
or no ice (open bars) after freezing at −10°C for 30 min. There were no
nematodes that had extracellular ice only. Data are means±s.e.m. (N=4,
replicate cultures). Different lowercase letters indicate significant differences
between means (P<0.05).
intestinal, muscle and epidermal cord cells of starved nematodes
(Fig. 3B). In contrast, the cytoplasm of fed nematodes is filled
with densely stained granular material, organelles and lipid
droplets (Fig. 3A).
In nematodes frozen at −10°C, processed by freeze substitution,
the former positions of ice are retained as lightly stained empty
spaces (Fig. 3C–F). The ice spaces were larger, fewer and less
regular in frozen starved Panagrolaimus sp. DAW1 (Fig. 3D) than
they were in frozen fed nematodes (Fig. 3C), and cellular structures
were much more disrupted in starved than in fed nematodes. Ice
spaces in P. redivivus were even larger and more irregular (Fig. 3E),
although these nematodes often also contained many small ice
spaces. The pattern and distribution of ice formed in P. murrayi was
quite variable but appeared intermediate between the pattern and
distribution in fed Panagrolaimus sp. and that seen in P. redivivus
and in starved Panagrolaimus sp. (Fig. 3F). Enlarged versions of
Fig. 3C–F are provided as Figs S1–S4.
In all three species, and in both fed and starved Panagrolaimus
sp. DAW1, most individuals contained intracellular ice (Fig. 4).
Percentage containing large ice crystals
RESEARCH ARTICLE
35
30
25
20
15
10
5
0
Panagrolaimus Panagrolaimus
fed
starved
P. murrayi
P. redivivus
Fig. 6. Percentage of Panagrolaimus sp. DAW1, Plectus murrayi and
Panagrellus redivivus that contained at least one large ice-filled space
(defined as being >10% of the total body area). The number of nematodes
measured is shown in Fig. 5.
There were no individuals that contained only extracellular ice,
although many of those that had intracellular ice also had
extracellular ice. In fed and starved Panagrolaimus sp., a small
percentage (7.5%) contained no ice and appeared dehydrated,
whereas in P. murrayi 19% contained no ice and 29% appeared
dehydrated. The dehydrated individuals contained either no ice or a
small percentage of ice (<10%). In P. redivivus there were no
individuals that did not contain ice and none appeared dehydrated
(Fig. 4). Overall, the proportion that contained intracellular ice was
significantly different between the four groups (F3,14=6.99,
P=0.007).
The five largest ice-filled spaces occupied the greatest proportion
of the body area in starved Panagrolaimus sp. and the smallest
proportion in P. murrayi (Fig. 5). Proportions between groups were
significantly different (F3,147=14.083, P<0.0001). The proportion
of nematodes that contained large ice-filled spaces (at least one that
was >10% of the body area) was greatest in starved Panagrolaimus
sp. and smallest in P. murrayi (Fig. 6). The largest ice-filled space
occupied 62.3% of the body area in starved Panagrolaimus sp.,
15.4% in fed Panagrolaimus sp., 26.4% in P. murrayi and 15.9% in
P. redivivus.
40
Percentage
30
b
a,b
20
a
c
10
0
Panagrolaimus Panagrolaimus
fed
starved
40
40
P. murrayi
P. redivivus
28
40
Fig. 5. Percentage of Panagrolaimus sp. DAW1, Plectus murrayi and
Panagrellus redivivus cross-sectional area occupied by the five largest
ice-filled spaces. The number shown below the x-axis labels is the number
of nematodes measured, after excluding those without ice. Data are means±
s.e.m. Different lowercase letters indicate significant differences between
means (P<0.05).
The survival of the nematode species and treatments tested after
exposure to freezing at −10°C is in line with that observed in previous
studies (Hayashi and Wharton, 2011; Raymond and Wharton, 2013;
Wharton and Raymond, 2015). This freezing exposure is survived
best by fed Panagrolaimus sp. DAW1. Survival is lower in starved
Panagrolaimus sp. and in P. murrayi, and there is very low survival in
P. redivivus. A further comparison of the cold tolerance of these
species is provided by calculations of the 50% survival temperatures
(S50) of nematodes exposed to a series of subzero temperatures under
the same conditions (not acclimated): Panagrolaimus sp., −25.4°C
(Smith et al., 2008); P. murrayi, −3.9°C (Wharton and Raymond,
2015); and P. redivivus, −1.3°C (Smith et al., 2008). The S50 of
starved Panagrolaimus sp. is −6.3°C (calculated from data from
Raymond and Wharton, 2013).
Freezing injuries may occur as a result of the disruption of cell
membranes. Vital dyes that assess cell viability based on membrane
integrity are often used to test the viability of spermatozoa and
oocytes preserved by freezing (Garner and Johnson, 1995). Vital
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Journal of Experimental Biology
DISCUSSION
dyes are useful for studying freezing injuries at the cellular level by
staining dissected tissues after cold exposure of insects (Yi and Lee,
2003). The use of vital dyes on whole nematodes enabled cell
damage after freezing to be visualised, and may be useful for future
studies on cellular injuries in nematodes, and similar animals,
generally. However, the technique is not applicable to all species.
Live P. redivivus showed limited staining, indicating that the dyes
were not entering the cells, and after freezing structural damage was
too extensive to distinguish stained nuclei. There is little cellular
damage after freezing in fed Panagrolaimus sp. More cellular
damage after freezing is apparent in P. murrayi and in starved
Panagrolaimus sp. This may be responsible for the lower survival of
these two groups of nematodes.
Freeze substitution preserves the location of ice as white spaces
(Ali and Wharton, 2014; Salinas-Flores et al., 2008; Wharton et al.,
2005), and confirms that for most of these nematodes freezing at
−10°C produces intracellular freezing. The pattern and distribution
of ice formed can thus be compared between a species that survives
intracellular freezing well (Panagrolaimus sp.), one that survives
poorly (P. redivivus) and one intermediate between these two
(P. murrayi). For Panagrolaimus sp., the pattern and distribution of
ice formed can also be seen when survival is compromised by
starvation. Starvation produces structural changes in unfrozen
controls, compared with that of fed Panagrolaimus sp., but it also
results in clear changes in the pattern and distribution of ice formed.
The pattern of ice formed in nematodes that survive intracellular
freezing at −10°C well (fed Panagrolaimus sp.) is that the ice
spaces are small and regular in size. In nematodes that show lower
levels of survival after intracellular freezing (starved Panagrolaimus
sp., P. murrayi and P. redivivus), at least some of the ice spaces are
much larger in size and less regular in shape. These are likely to have
caused the structural damage to cells, as seen in starved
Panagrolaimus sp. by staining with vital dyes.
Plectus murrayi shows a different pattern and distribution of ice
formed. There were more individuals with no ice formed at −10°C
and with a shrunken appearance than was observed in the other
species. Those with no ice are surviving by cryoprotective
dehydration; this is also indicated by the shrinkage observed. The
majority of P. murrayi, however, do freeze, and this is the case even
if freezing occurs at −1°C (Wharton and Raymond, 2015). Many of
those that freeze at −10°C are killed, but a proportion survives.
Frozen P. murrayi contain on average smaller ice spaces than do
P. redivivus and starved Panagrolaimus sp.; those individuals with
smaller ice crystals and a smaller proportion of ice are likely to be
the ones that survive. Oyster oocytes (Crassostrea gigas) can
survive intracellular freezing if the ice spaces are small (SalinasFlores et al., 2008). Survival of intracellular freezing could be more
common amongst cells and tissues frozen for cryopreservation than
is currently realised, as the formation of small ice crystals is not
observed using optical cryomicroscopy (Salinas-Flores et al., 2008).
Ice has also been observed in the entomopathogenic nematode
Steinernema feltiae (Ali and Wharton, 2014). This nematode
survives intracellular freezing but only if freezing occurs at a
relatively high subzero temperature (down to −3°C). Small ice
spaces are found in S. feltiae frozen at −1°C or −3°C and the
nematodes survive, but large ice spaces are found in those frozen at
−10°C and the nematodes do not survive.
Control of the size of the ice spaces thus appears to be important
for the survival of intracellular freezing in nematodes. What could
be controlling this? The trehalose concentration of Panagrolaimus
sp. increases upon acclimation at low temperatures (Wharton et al.,
2000); genes involved in trehalose synthesis have been identified in
2064
Journal of Experimental Biology (2016) 219, 2060-2065 doi:10.1242/jeb.137190
the transcriptome of this nematode (Thorne et al., 2014) and are
upregulated during cold acclimation (A. Seybold, Molecular
adaptation mechanisms in the Antarctic nematode Panagrolaimus
davidi, PhD thesis, University of Otago, 2015). However, trehalose
concentrations are still relatively low compared with those found in
other cold-tolerant organisms.
Panagrolaimus sp. produces an ice active substance with strong
recrystallization inhibition activity (Ramløv et al., 1996; Smith
et al., 2008). In a study comparing the freezing survival and
recrystallization inhibition of six nematode species (Smith et al.,
2008), P. davidi (=Panagrolaimus sp. DAW1) had the highest rates
of survival and levels of recrystallization inhibition activity,
whilst P. redivivus had lower survival and the lowest level of
recrystallization inhibition activity. The freezing survival of
P. murrayi is intermediate between these two species and has
only weak recrystallization inhibition activity (Wharton and
Raymond, 2015). Several other Panagrolaimus species and
strains can survive freezing, and extracts of these also inhibit
recrystallization in a splat freezing assay and the growth of single
crystals in a nanolitre osmometer (McGill et al., 2015). It seems
likely that it is an ice active substance with recrystallization
inhibition activity that is responsible for controlling the size of the
ice spaces and that this is an important adaptation for the ability of
some nematodes to survive intracellular freezing.
Acknowledgements
We would like to thank Karen Judge and Matthew Downes for technical assistance
and the Otago Centre for Electron Microscopy for access to their facilities.
Competing interests
The authors declare no competing or financial interests.
Author contributions
M.R.R. and D.A.W. designed the study, M.R.R. conducted the experiments, M.R.R.
and D.A.W. analysed the data, and D.A.W. and M.R.R. wrote the paper.
Funding
M.R.R. was supported by a Kelly Tarlton’s Antarctica New Zealand Scholarship and
the Fanny Evans Postgraduate Scholarship for Women. Funding for this study was
also provided by the University of Otago (PBRF funds).
Supplementary information
Supplementary information available online at
http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.137190/-/DC1
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